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Page de résumé pour ULgetd-05102010-154624

Auteur : Lassaux, Patricia
E-mail de l'auteur : plassaux@ulg.ac.be
URN : ULgetd-05102010-154624
Langue : Français/French
Titre : Caractérisation biochimique et structurale de la métallo-β-lactamase VIM-4. Sélection de nouveaux inhibiteurs de métallo-β-lactamases.
Intitulé du diplôme : Doctorat en sciences
Département : FS - Département des sciences de la vie
Jury :
Nom : Titre :
Charlier, P. Membre du jury/Committee Member
Damblon, C. Membre du jury/Committee Member
Dehareng, D. Membre du jury/Committee Member
Ferrer, J.L. Membre du jury/Committee Member
Lins, L. Membre du jury/Committee Member
Spencer, J. Membre du jury/Committee Member
De Pauw, E. Président du jury/Committee Chair
Galleni, M. Promoteur/Director
Mots-clés :
  • antibacterial resistance/ résistance aux antibiotiques
  • apoenzyme/apoenzyme
  • inhibition/inhibition
  • enzymatic kinetics/cinétique enzymatique
  • crystallograhic structure/structure crystallographique
Date de soutenance : 2010-06-04
Type d'accès : Restreint/Intranet
Résumé :

The Pseudomonas aeruginosa VIM-4 metallo-β-lactamase is a member of the subclass B1. It belongs to the VIM-type β-lactamases encoded by genetic mobile element such as class 1 integrons. This particularity enhances the relevance of VIM type enzymes since these enzymes are now disseminated among important nosocomial strains such as Klebsiella pneumoniae and Pseudomonas aeruginosa. In consequence, the study of VIM-4 was performed to determine its kinetic parameters, its 3D structure and the dependence of its activity on Zn2+ concentration. VIM-4 has been described as an efficient enzyme able to hydrolyze all the β-lactam compounds. We observed that VIM-4 activity is dependent on the Zn2+ concentration in the buffer, with a maximal activity obtained at 50 µM. Two different forms of VIM-4 were observed by X-ray crystallography in the presence or absence of Zn2+ in the crystallogenesis buffer. The first form contains two Zn2+ and the second possesses only one Zn2+ in the histidine site. The apoenzyme form was obtained. Its study revealed that this form was poorly stable with a less structured shape. The active form is not restored even in presence of a large Zn2+ excess. In order to determine the Zn2+ stoechiometry in VIM-4, ICPMS (Inductively coupled plasma mass spectrometry) experiments were performed. The results of this study indicated that the mono-zinc form of VIM-4 is favored in absence of Zn2+ ions in the buffer and the di-zinc form appears in the presence of added Zn2+ ions. The dissociation constants of the Zn2+ ions with VIM-4 enzyme were determined using benzylpenicillin. The dissociation constants of the first, the second and the third Zn2+ are respectively KD1 which is clearly below 1 µM, KD2 equal to 8.5 µM and KD3 within a range of 200 to 500 µM. A complementary titration of the free Zn2+ ions from denatured VIM-4 with a chromophore chelating agent seems to confirm this feature as only one Zn2+ could be titrated. Then with no added Zn2+, VIM-4 would be in a mono-zinc form and with a Zn2+ concentration higher than 10 µM, the di-zinc form of VIM-4 would be present.

In the last decade, numerous studies have reported an increasing mortality among patients in intensive care due to multiresistant pathogens. These strains are producing at least two different classes of β-lactamases. In the eighties, a strategy of combination of β-lactamase inactivator and β-lactam antibiotics was developed against serine β-lactamases. Some of the members of this family have already developed variants resistant to these inhibitors; however this combination remains efficient. In the case of the MBLs, extensive studies have been performed to find a generic inhibitor. Unfortunately, due to the heterogeneity of this enzyme group, no solution has been found. The development of a metallo-β-lactamase inhibitor, particularly active against acquired MBLs, which are the most relevant enzymes, is thus needed. Our second purpose was the identification of new molecules that could be developed as broad spectrum MBLs inhibitors. We found a new class of compounds, mercaptophosphonates derivatives, which can be used as leads for finding generic MBL inhibitors.

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